The present invention generally relates to compositions and methods for the administration of particulates comprising at least one bioactive agent which, in selected embodiments, may comprise an immunoactive agent. In this respect, the invention provides for both topical and systemic delivery of the bioactive agent using, for example, the respiratory, gastrointestinal or urogenital tracts. In particularly preferred embodiments, the disclosed compositions will be used in conjunction with inhalation devices such as metered dose inhalers, dry powder inhalers, atomizers or nebulizers for targeted delivery to mucosal surfaces.
Vertebrates possess the ability to mount an immune response as a defense against pathogens from the environment as well as against aberrant cells, such as tumor cells, which develop internally. This can take the form of innate or passive immunity, which is mediated by neutrophils and cells of the monocyte/macrophage lineage, or the form of acquired or active immunity mediated by lymphocytes against a specific antigenic sequence. Active immune responses can themselves be further subdivided into two arms, the humoral response which entails the production of specific antibodies which serve to neutralize antigens exposed to the systemic circulation and aid in their uptake by professional phagocytic cells, and the cellular arm which is required for recognition of infected or aberrant cells within the body.
In both cases the specific response is triggered by the intracellular processing of antigen. When the antigen is processed through the cytoplasmic route, the resultant peptides are bound to nascent MHC class I molecules which facilitates appropriate presentation to effector T-cells. MHC class I presentation favors recognition by cytotoxic T lymphocytes. In contrast, intracellular processing via the endocytic route results in presentation on MHC class II molecules which favors T helper responses involved in stimulation of the humoral arm. The goal of vaccination is to prime both responses and generate memory T cells, such that the immune system is primed to react to a pathogenic infection. Such a response is promoted by the coadministration of signals that promote costimulatory molecule expression, so called xe2x80x9cadjuvants.xe2x80x9d Engagement of both the humoral and cellular immune responses leads to broad based immunity and is the preferred goal for intracellular pathogens. The absence of appropriate costimulatory molecule expression can lead to a state of T cell unresponsiveness.
In this regard, modulation of an immune response can take one of two directions; either to elicit an immune response directed against a foreign pathogenic agent or antigen thereof, or to suppress an inappropriate reaction mounted against a self-epitope that leads to chronic inflammation. Such chronic reactions against self-epitopes are associated with various autoimmune diseases such as diabetes, typically type I, multiple sclerosis, rheumatoid arthritis or lupus erythrematosis. In either case, the active agent frequently takes the form of a relatively complex peptide, protein, RNA or DNA-based entity or other macromolecular structure rather than small chemical entities typical of classical pharmaceutical agents. These complex bioactive agents generally exhibit poor bioavailability when administered orally, and therefore have traditionally been administered by invasive parenteral injection. Recently however, it has been suggested that relatively large biomolecules may be delivered via mucosal routes, e.g. by inhalation. Delivery of these agents into systemic circulation through inhalation is particularly attractive since administration via the respiratory mucosa bypasses the digestive enzymes of the GI tract. Furthermore, it offers the potential for increased bioavailability for peptides and proteins because of the large surface area available for exchange with systemic circulation. While the molecular weight cut-off for oral bioavailability is generally regarded to be in the range of 500 Daltons, peptide hormones or analogues of larger molecular weight (e.g., 1.8 kD desmopressin, 5.8 kD insulin, 9.5 kD parathyroid hormone), have been shown to be absorbed across the nasal or pulmonary mucosa intact into the systemic circulation.
Besides allowing for the effective delivery of protein, peptide, viral and DNA formulations without degradation, targeted delivery to the mucosal surface itself may offer a benefit if it elicits a local immune response within the MALT (mucosa-associated lymphoid tissue) lymphoid system. Mucosal vaccination is of particular interest for vaccines designed against pathogens whose port of entry is typically at one of the mucosal surfaces interfacing the body with the external environment. The MALT lymphoid system resides within the lamina propria of the mucosa. When foreign antigen is presented to local dendritic cells, there is a local amplification and maturation of B-cell precursors, which produce IgA and IgM antibodies in addition to the IgG antibodies typically induced by systemic delivery of antigen. The former are secreted through specialized transport receptors by a process known as transcytosis across the mucosal surface into the lumen. There, they provide a first line of defense against invading pathogens at the mucosal surface. Recent evidence indicates that, in addition to binding pathogenic antigens, the resultant formation of immune complexes may in and of itself inhibit viral transmission occurring via the transcytotic route. By priming this first line immune response to antigens derived from pathogens, mucosal immunization should greatly enhance the efficiency with which the organism first intercepts an invading pathogen.
Several previous attempts have been made to exploit this uptake mechanism and provide for the effective delivery of peptides or proteins. For example, U.S. Pat. No. 5,756,104 describes the use of liposome formulations for intranasal vaccine formulations. These formulations appear to comprise aqueous carriers having liposomes and free antigenic material dispersed therein. While the compositions were found to elicit an immune response, they appear to be extremely labile and susceptible to degradation over time. In a practical sense this is a substantial drawback.
Attempts to overcome such limitations and further increase delivery efficiency have resulted in the development of dry powders for the administration of relatively large biomolecules. Unfortunately, conventional powdered preparations (i.e. micronized) often fail to provide accurate, reproducible dosing over extended periods. In part, this is because the powders tend to aggregate due to hydrophobic or electrostatic interactions between the fine particles. Such cohesion may be partially overcome through the use of larger carrier particles (i.e. lactose) to inhibit aggregation. However, these larger particles and associated drug often fail to reach the targeted cells resulting in uneven delivery profiles. Further, crude mixtures comprising carrier molecules provide little, if any, protection for the incorporated biomolecule. Accordingly, as with the aqueous compositions described above, such preparations are subject to degradation and loss of activity over time.
More recently, improved formulation methods have been undertaken in order to overcome the limitations associated with conventional prior art powders and aqueous preparations. In this regard, U.S. patent applications Ser. Nos. 09/218,209 and 09/219,736, incorporated herein by reference, describe methods and processes for generating preparations comprising bioactive agents in microparticulate form. The resultant powders, which preferably exhibit a hollow, porous morphology, are suitable for use in inhalation devices such as dry powder inhalers (DPIs) or, when suspended in a nonaqueous liquid (i.e. a hydrofluoroalkane or fluorocarbon), metered dose inhalers (MDIs) and nebulizers. Moreover, the mild conditions used during the formulation process support retention of biological activity making the preparations particularly compatible for use with proteins and peptides as well as more complex macromolecular structures such as viruses. Additionally, since the resultant powders have very low residual water content, which can be further maintained by formulation in short-chain fluorocarbons or fluorochemicals such as propellants or the longer chain fluorochemicals such as perfluorooctyl bromide (PFOB), these formulations provide a stable means for storage of labile bioactive agents.
Besides enhanced stability, the preferred hollow, porous morphology of the microparticulates provides aerodynamic characteristics that are particularly compatible with inhalation therapies. Further, the particulate characteristics allows for the formation of exceptionally stable dispersions and makes them especially compatible with hydrofluoroalkane propellants such as HFA-134a as well as other fluorocarbon liquid vehicles like PFOB. Thus, whether used in a dry form or as a nonaqueous dispersion, the microparticulates provide for good dose reproducibility, excellent plume characteristics (a measure of the uniformity of a propellant or dry powder spray) and a high percentage of the dose delivered as the respirable fraction (as opposed to deposition in the device or throat). These properties suggest that the disclosed microparticles offer substantial theoretical advantages as far as delivery deep into the lung. Such deep deposition is preferred where delivery into the systemic circulation is desired since uptake of large macromolecules like proteins and peptides is optimal at the level of the alveoli.
While the use of such microparticulate preparations is a substantial improvement over conventional prior art delivery methods, there still remains a need to provide for the targeted delivery of bioactive, immunomodulating or immunoactive agents that results in an enhanced physiological response.
Accordingly, it is an object of the present invention to provide compositions, systems and methods that provide for the generation of an enhanced immune response.
It is another object to provide for the effective delivery of immunoactive agents, including vaccines and immunomodulating agents, to the mucosal surfaces of a patient in need thereof.
It is yet a further object of the present invention to provide vaccine or other bioactive formulations that do not require refrigeration or freezing to maintain activity.
It is still a further object of the present invention to provide for the establishment of passive and active immunity via inhalation therapies.
It is yet another object of the present invention to provide for stable preparations of immunoactive agents that may be used to confer immunity or down regulate the immune system of a patient in need thereof.
These and other objects are provided for by the invention disclosed and claimed herein. To that end, the methods and associated compositions of the present invention allow, in a broad aspect, for the improved delivery of bioactive agents to selected target sites in a powdered or particulate form. More particularly, it has been surprisingly been found that the disclosed methods and compositions may be used to enhance or increase the activity of an incorporated bioactive agent, which preferably comprises an immunoactive agent, following administration. In this regard, the vaccines of the instant invention appear to exhibit an xe2x80x9cadjuvant effectxe2x80x9d that may provoke an enhanced immune response an order of magnitude or more greater than that provoked by a comparable prior art vaccine formulation. Besides this unexpected improvement in potency, relatively gentle formulation techniques may be combined with particulate morphology and composition to protect and enhance the activity of any incorporated agents. This allows for the formation of relatively efficacious preparations that retain their biological activity without the need for refrigeration or freezing. Further, unlike prior art powders or dispersions for drug delivery, the present invention preferably employs novel techniques to reduce attractive forces between the particles, resulting in improved flowability and dispersibility. When these powders are incorporated in a nonaqueous suspension medium (e.g. a liquid fluorochemical) these same characteristics provide for reduced flocculation, sedimentation or creaming that may further reduce the rate of agent degradation. Finally, administration of the disclosed particulates or dispersions to selected target sites such as mucosal surfaces may further serve to optimize or enhance bioactivity. As such, the dispersions or powders of the present invention may be used to effectively deliver bioactive agents in conjunction with metered dose inhalers, dry powder inhalers, atomizers, aerosolizers, nasal pumps, spray bottles, nebulizers or liquid dose instillation (LDI) techniques.
A particularly beneficial feature of the disclosed particulate formulation technology is that a wide range of bioactive structures can be incorporated in the stabilized dispersions or powders irrespective of their hydrophobicity or hydrophilicity. In preferred embodiments, the bioactive powders will be produced using relatively mild spray drying methodology. Due to such compatible particulate formulation techniques, larger, more labile biomolecules such as peptides, proteins or genetic material may readily be incorporated in the disclosed compositions without adverse effects or undue loss of activity. These same formulation techniques and resulting particulates further provide for the incorporation and delivery of relatively high doses (ca. 10 mg) of bioactive agents using conventional administration techniques and systems. Thus, whether administered in the form of a dry powder or stabilized dispersion, the novel particulate fabrication techniques and enhanced response afforded by the disclosed preparations lead to the effective delivery of bioactive agents to targeted sites such as the mucosa.
In connection with the present invention, the term xe2x80x9cbioactive agentxe2x80x9d refers to any active peptide or protein, such as a hormone, cytokine or chemokine or an immunoactive agent. That is, while the disclosed compositions and methods are compatible with almost any bioactive agent, they have been discovered to be surprisingly effective for the delivery or administration of immunoactive agents designed to modulate immune responses such as, for example, eliciting an immune response to a foreign antigen or pathogen or down regulating an active immune reaction. Accordingly, as used herein, the terms xe2x80x9cimmunoactive agents,xe2x80x9d or xe2x80x9cimmunologically active agents,xe2x80x9d will comprise any molecule that may be used to elicit a physiological or immune response or modulate pre-existing responses in a subject. Such immunoactive agents or biologics may comprise peptides, polypeptides, proteins, carbohydrates, genetic material including DNA, RNA and antisense constructs, as well as microbes including viruses, phages and bacteria.
In addition, molecules that may function as cofactors, potentiators or penetration enhancers can be readily co-formulated in the particulates described herein. Those skilled in the art will appreciate that any compound which acts to improve the uptake, presentation or bioavailability may function as a potentiator or penetration enhancer in accordance with the teachings herein. For instance, compounds that can alter or increase the membrane permeability of a cell may function as potentiators or penetration enhancers. Exemplary potentiators or penetration enhancers may include chelating agents (e.g. EDTA, citric acid), detergents or surfactants (e.g. 9-lauryl ether), fatty acids (e.g. oleic acid) and bile salts (e.g. sodium glycocholate). Particularly preferred penetration enhancers comprise relatively short chain phospholipids having chain lengths of less than about 10 carbons. As with the bioactive agents, and as will be discussed in more detail below, the selected potentiators or penetration enhancers may be incorporated in, or associated with, particulates in varying concentrations.
With regard to the particulates, microparticulates or perforated microstructures of the present invention, those skilled in the art will appreciate that they may be formed of any biocompatible material providing the desired physical characteristics or morphology. In this respect, perforated microstructures will preferably comprise pores, voids, defects or other interstitial spaces that act to reduce attractive forces by minimizing surface interactions and decreasing shear forces. This morphology acts to reduce aggregation and improve dispersability. Yet, given these constraints, it will be appreciated that any biocompatible material or configuration may be used to form the microstructure matrix. As to the selected materials, it is desirable that the microstructure incorporates at least one surfactant which, in preferred embodiments, will act as a penetration enhancer. Preferably, this surfactant will comprise a phospholipid or other surfactant or amphiphile approved for pharmaceutical use. Similarly, it is preferred that the microstructures incorporate at least one bioactive agent or biologic. As to the configuration, selected embodiments of the invention comprise spray dried, hollow microspheres having a relatively thin porous wall defining a large internal void, although, other void containing or perforated structures are contemplated as well.
It has unexpectedly been found that the use of hollow and/or porous perforated microstructures may substantially reduce attractive molecular forces, such as van der Waals forces, which dominate prior art powdered preparations and dispersions. In this respect, the powdered compositions typically have relatively low bulk densities that contribute to the flowability of the preparations while providing the desired characteristics for inhalation therapies. More particularly, the use of relatively low density perforated (or porous) microstructures or microparticulates significantly reduces attractive forces between the particles thereby lowering the shear forces required to achieve flowability of the resulting powders. The relatively low density of the perforated microstructures also provides for superior aerodynamic performance when used in inhalation therapy. In dispersions, the physical characteristics of these powders provide for the formation of stable preparations. Moreover, by selecting dispersion components in accordance with the teachings herein, interparticle attractive forces may further be reduced to provide formulations or preparations having enhanced stability.
While preferred embodiments of the invention comprise perforated microstructures or porous particulates, relatively nonporous or solid particulates may also be used to prepare powders or dispersions that are compatible with the teachings herein. That is, powders or dispersions comprising suspensions of relatively nonporous or solid particulates are also contemplated as being within the scope of the present invention. In this respect, such relatively nonporous particulates may comprise micronized particles, milled particles or nanocrystals. Accordingly, as used herein the term xe2x80x9cparticulatexe2x80x9d shall be interpreted broadly and held to comprise particles of any porosity and or density, including both perforated microstructures and relatively nonporous particles.
As previously alluded to, the disclosed powders may be dispersed in an appropriate nonaqueous suspension medium to provide stabilized dispersions comprising a selected bioactive agent. Such dispersions are particularly useful in metered dose inhalers, atomizers nasal pumps, spray bottles and nebulizers. Other embodiments of the invention comprise stabilized dispersions that may be administered directly to the lung or nasal cavity using direct instillation techniques. In any case, particularly preferred suspension mediums comprise fluorochemicals (i.e. perfluorocarbons or fluorocarbons) that are liquid at room temperature or fluorinated propellants (i.e. hydrofluoroalkanes or chlorofluorocarbons). Because of their beneficial wetting characteristics, some fluorochemicals may be able to provide for the dispersion of particles deeper into the lung or other mucosal surface, thereby improving systemic delivery. Moreover, such suspension media tend to be anhydrous thereby retarding hydrolytic degradation of the incorporated bioactive agents. Finally, fluorochemicals are generally bacteriostatic thus decreasing the potential for microbial growth and associated proteolytic decay in compatible preparations.
With regard to the delivery of the disclosed powders or stabilized dispersions, another aspect of the present invention is directed to inhalation systems for the administration of one or more bioactive agents or biologics to a patient. As alluded to above, exemplary inhalation devices compatible with the present invention may comprise an atomizer, a nasal pump, a sprayer or spray bottle, a dry powder inhaler, a metered dose inhaler or a nebulizer. In preferred embodiments, these inhalation systems will deliver the bioactive agent to the desired physiological site (e.g. a mucosal surface) as an aerosol. For the purposes of the instant application the term xe2x80x9caerosolizedxe2x80x9d shall be held to mean a gaseous suspension of fine solid or liquid particles unless otherwise dictated by contextual restraints. That is, an aerosol or aerosolized medicament may be generated, for example, by a dry powder inhaler, a metered dose inhaler, an atomizer, a spray bottle or a nebulizer. Of course, as explained in more detail below, the compositions of the present invention may also be delivered directly (e.g. by conventional injection or needleless injection) or using such techniques as liquid dose instillation. In especially preferred embodiments the compositions of the present invention are contacted with a mucosal surface (e.g. via inhalation) to elicit both mucosal and systemic immunity.
While the powders or stabilized dispersions of the present invention are particularly suitable for administration of bioactive agents to mucosal surfaces, it will be appreciated that they may also be used for the localized or systemic administration of compounds to any location of the body. Accordingly, it should be emphasized that, in preferred embodiments, the formulations may be administered using a number of different routes including, but not limited to, the gastrointestinal tract, the respiratory tract, topically, intramuscularly, parenterally, intradermally, transdermally, intraperitoneally, nasally, vaginally, rectally, aurally, buccally, orally or ocularly. In this respect those skilled in the art will appreciate that the selected route of administration will largely be determined by the choice of bioactive agent and the desired response of the subject.
Other objects, features and advantages of the present invention will be apparent to those skilled in the art from a consideration of the following detailed description of preferred exemplary embodiments thereof.